Formulation for stabilizing proteins, which is free of mammalian excipient

ABSTRACT

The present invention pertains to a formulation comprising a hydrophilic polymer, a mixture of a polyalcohol and a sugar, wherein the weight ratio of polyalcohol to sugar is between 2:1 to 5:1 (wt-%), a detergent, wherein the formulation is free of stabilising proteins.

FIELD OF THE INVENTION

The present invention pertains to a formulation for stabilizingproteins, which is free of mammalian excipients. In particular, itpertains to a formulation comprising a hydrophilic polymer, a mixture ofa polyalcohol and a sugar, wherein the weight ratio of polyalcohol tosugar is from 2:1 to 5:1 (wt-%), a detergent, and wherein theformulation is free of stabilising proteins. In one embodiment, thepresent invention pertains to a kit, wherein said kit comprises one ormore containers comprising the said formulation/composition,instructions for use and optionally, a pharmaceutically acceptablesterile solvent.

BACKGROUND OF THE INVENTION

Protein formulations, which are free of stabilizing proteins are knownin the art. WO 2006/020208 relates to pharmaceutical compositionscomprising botulinum toxin and a non-proteinaceous stabilizing agent,which retains the activity of the botulinum toxin in an aqueoussolution.

WO 2006/005910 relates to solid or liquid pharmaceutical compositionscomprising Botulinum toxin complex or high purity botulinum toxin and asurfactant. A maximum of six months stability at 23° C. to 27° C. isreported therein.

WO 2007/041664 relates to a pharmaceutical composition comprising abotulinum toxin and a polyvinylpyrollidone (PVP) and optionally adisaccharide.

WO 2004/006954 relates to a pharmaceutical composition comprising astabilized botulinum toxin and at least one enhancing agent forfacilitating transdermal delivery of the botulinum toxin into a humanpatient by enhancing the permeability of the patient's skin.

WO 01/58472 discloses a pharmaceutical composition suitable forinjection into a human patient, comprising a botulinum toxin and apolysaccharide. It also discloses a pharmaceutical compositioncomprising a neurotoxin and hydroxyethyl starch.

WO 2006/079722 relates to the use of liquid compositions forimplementing the method of freeze-drying proteins, to stabilize saidproteins, said compositions comprising; a filler agent having a collapsetemperature between −18° C. and 0° C., a stabilizer, a buffer solution,and, as the case may be, a nonionic surfactant.

OBJECTS OF THE INVENTION

An object of the present invention was to provide a novel formulationfor stabilizing proteins, which is free of stabilizing proteins. Suchformulations may be formulated such that they provide superior stabilityto proteins, compared to formulations of the prior art.

This and other objects were achieved by the formulation being thesubject of this application.

SUMMARY OF THE INVENTION

The objects of the invention were achieved by the formulation beingsubject of this application. The present invention pertains to aformulation comprising a hydrophilic polymer, a mixture of a polyalcoholand a sugar, wherein the weight ratio of polyalcohol to sugar is from(2:1) to (5:1) (wt-%), e.g. (2:1), (2.5:1), (3:1), (3.5:1), (4:1),(4.5:1), (5:1), a detergent, and wherein the formulation is free ofstabilising proteins. In one further embodiment, the present inventionpertains to a composition that comprises the said formulation and apeptide, a protein or a mixture thereof, naturally occurring ormodified/artificial.

In one further embodiment the composition of the invention islyophilised.

A further aspect of the present invention relates to a compositioncomprising a peptide or a protein, or a mixture thereof, as definedherein, for use as a medicament, a cosmetic product, a cosmeceuticalproduct or a diagnostic product.

One further aspect of the present invention relates to said compositionfor use in the treatment of a disease or condition caused by orassociated with hyperactive cholinergic innervation of muscles orexocrine glands in a patient.

A further aspect of the present invention relates to a kit comprisingone or more of a container comprising said formulation/composition andinstructions for use of the said formulation and optionally apharmaceutically acceptable sterile solvent.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to a formulation comprising a hydrophilicpolymer, a mixture of a polyalcohol and a sugar, wherein the weightratio of polyalcohol to sugar is between (2:1) to (5:1) (wt-%), e.g.(2:1), (2.5:1), (3:1), (3.5:1), (4:1), (4.5:1), (5:1), a detergent, andwherein said formulation is free of stabilising proteins.

The term “formulation” as used herein relates to a mixture comprisingpharmaceutically acceptable excipients and encompasses liquid, solid,semisolid, colloidal and all other forms known to the person skilled inthe art. The said formulation herein is free of stabilizing proteins.

The term “composition” as used in the instant invention relates to aformulation as claimed herein which further comprises a peptide, aprotein or a mixture thereof.

The formulation of the invention comprises a hydrophilic polymer.

The term “polymer” as used herein relates to structures composed ofrepeating units. The term “polymer” within the scope of the instantinvention is employed both for homopolymers and copolymers.

The term “hydrophilic” as used herein relates to substances, materials,excipients or pharmaceutically active ingredients which are wettable bywater.

In one embodiment of the present invention, the hydrophilic polymer isselected from the group consisting of hyaluronic acid,polyvinylpyrollidone (PVP), copolymers of N-vinylpyrollidone, cellulosederivatives, Polyethyleneglycol (PEG), PEG/PPG block copolymers, homo-and copolymers of acrylic and methacrylic acid, polyurethanes, polyvinylalcohol (PVA), polyvinylethers, maleic anhydride based copolymers,polyesters, vinylamines, polyethyleneimines, polyethyleneoxides,poly(carboxylic acids), polyamides, polyanhydrides, polyphosphazenes andmixtures thereof.

The said cellulose derivative may be selected from the group consistingof hydroxypropyl methyl cellulose, hydroxypropyl cellulose, hydroxyethylcellulose, methyl cellulose, carboxymethyl cellulose, dextran, andmixtures thereof.

The term “polyvinylpyrrolidone” as used herein refers to a water-solublepolymer made from the monomer N-vinylpyrrolidone. The terms andabbreviations “PVP, povidone, polyvidone, crospovidone, Kollidone” areused synonymously.

The said polyvinylpyrollidone (PVP) may be Kollidon 12 PF, Kollidon 17PF, Kollidon 25, Kollidon 30, Kollidon 90 F, povidone, crospovidone,Kollidon VA 64 and copovidone or a mixture thereof.

The term “hyaluronic acid” within the meaning of the instant inventionrefers to a non-sulfated glycosaminoglycan. In one embodiment thehyaluronic acid has a molecular weight of 0.8 to 1.2×10⁶ Da.Furthermore, within the present invention also crosslinked hyaluronicacid may be used. The term “hyaluronic acid” is used synonymously withthe term “hyaluronan”. Within the present invention the term “hyaluronicacid” also encompasses derivatives of hyaluronic acid, such as saltsthereof, e.g. sodium, potassium, magnesium and calcium salts. Furtherthe term “hyaluronic acid” encompasses all natural and syntheticderivates thereof. It is a molecule having typically a molecular weightof 10 kDa and 4.5×10⁶ Da.

The formulation of the invention comprises a mixture of polyalcohol andsugar in a weight ratio of from (2:1) to (5:1) (wt. %).

The term “polyalcohol” as used herein relates to a group ofcarbohydrate-based ingredients, which are employed to protect theprotein against instability. The term “polyol” and “sugar alcohols” areused synonymously.

The term “sugar” as used herein relates to any monosaccharide,disaccharide and polysaccharide. The term “monosaccharide” as usedherein relates to the basic units of carbohydrates. The term“disaccharide” within the scope of the present invention relates tocarbohydrates composed of two monosaccharides. The term“polysaccharides” as used herein relates to repeating units ofmonosaccharides, wherein the monosaccharides are bound with glycosidicbonds.

The term “mixture” as used herein relates to compositions of homogeneousor heterogeneous nature, wherein at least two substances of the same ordifferent composite or structure are mixed by employing the methods anddevices known to the person skilled in the art. The term “mixture”within the scope of the instant invention encompasses mixtures in solid,liquid and semisolid form.

The term “mixing” as used herein relates to combining at least twoactive or inactive ingredients at various proportions. Mixing relates toany process or action which combines also at least two different activeor inactive substances from the same group or from different groups, inany sequential order. The term “mixing” also discloses any process oraction which combines any active ingredient with any excipient.

In one embodiment of the present invention the polyalcohol is selectedfrom the group consisting of mannitol, inositol, lactilol, isomalt,xylitol, erythritol and sorbitol.

In one further embodiment of the present invention the sugar is selectedfrom the group consisting of monosaccharides, wherein saidmonosaccharides may be glucose, thioglucose, thiomannose, thiofructose,fructose and galactose. In another embodiment the sugar is adisaccharide, wherein said disaccharide may be trehalose, sucrose,octa-O-acetyl-thiotrehalose, thiosucrose, thiomaltose, maltose, andmaltitol. In one further embodiment the sugar is a polysaccharide,wherein said polysaccharide may be an alginate, hydroxyethyl starch andhydroxypropyl starch.

The present invention claims a formulation, wherein a polyalcohol and asugar are mixed to obtain a mixture of polyalcohol and sugar at a weightratio of (2:1) to (5:1). In one further embodiment said mixture ofpolyalcohol and sugar is at a weight ratio of (2:1) to (3:1), e.g.(2:1), (2.5:1), (3:1). In another embodiment said mixture of polyalcoholand sugar is at a weight ratio of (3:1).

According to one embodiment of the instant invention the mixture ofpolyalcohol and sugar comprises mannitol and sucrose at a weight rationof (2:1) to (5:1), e.g. (2:1), (2.5:1), (3:1), (3.5:1), (4:1), (4.5:1),(5:1). In one further embodiment of the instant invention the mixture ofpolyalcohol and sugar comprises mannitol and sucrose at a weight rationof (3:1).

Said polyalcohol and said sugar may be mixed by using V-blenders (twinshell blenders), rotary drum mixers, double ribbon blenders, plowmixers, paddle mixers, double cone blenders. The skilled person will beable to select the correct mixer depending on bench-top scale or highscale. The mixing time will depend on the batch size, quality ofexcipients, e.g. particle size of the powder and the mixer type.

The formulation of the invention also comprises a detergent.

The term “detergent” as used herein relates to any substance employed tosolubilize or stabilize another substance, which may be either apharmaceutical active ingredient or another excipient in a formulation.Said detergent may stabilize said protein or peptide either stericallyor electrostatically. The term “detergent” is used synonymously with theterms “surfactants” or “surface active agents”.

In one embodiment of the present invention the detergent is selectedfrom the group consisting of non-ionic surfactants.

The term “non-ionic surfactants” within the meaning of the instantinvention refers to surfactants having no positive or negative charge.

According to one aspect said non-ionic surfactants may be sorbitanesters (sorbitan monolaurate, sorbitan monopalmitate, sorbitanmonostearate, sorbitan tristearate, sorbitan monooleate, Sorbitantrioleate), polysorbates (polyoxyethylene (20) sorbitan monolaurate(Polysorbate 20), polyoxyethylene (20) sorbitan monopalmitate,polyoxyethylene (20) Sorbitan monostearate, polyoxyethylene (20)sorbitan tristearate, polyoxyethylene (20) Sorbitan trioleate,Polyoxyethylen(20)-sorbitan-monooleate (Tween 80/Polysorbate 80)),poloxamers (poloxamer 407, poloxamer 188), cremophor, and mixturethereof.

In another embodiment said detergent is anionic surfactant.

The term “anionic surfactant” within the meaning of the presentinvention refers to surfactants comprising an anionic hydrophilic group.

According to one aspect said anionic surfactant may betetradecyltrimethylammonium bromide, dodecyltrimethylammonium bromide,sodium laureth sulphate, sodium dodecyl sulphate (SDS), cetrimide,hexadecyltrimethylammonium bromide, and a mixture thereof.

In one further embodiment said detergent is a cationic surfactant.

The term “cationic surfactant” within the meaning of the instantinvention encompasses surfactants comprising a cationic hydrophilicgroup.

According to one aspect said cationic surfactant may be benzalkoniumchloride, cetyl trimethlammonium bromide (CTAB), cetylpyridiniumchloride (CPC), benzethonium chloride (BZT), and mixtures thereof.

In one embodiment of the present invention the concentration of thedetergent is not more than 0.5 mg/g based on the total weight of theproduction bulk composition, i.e. the total amount of the formulation ofthe invention, the peptide or protein to be stabilized and the sterilesolvent added for injection, typically water or an isotonic salinesolution. In one further embodiment of the instant invention, theconcentration of the detergent is between 0.1 mg/g and 0.3 mg/g based onthe total weight of the production bulk composition. In anotherembodiment of the instant invention the detergent employed isPolysorbate 80 and the concentration thereof is 0.2 mg/g based on thetotal weight of the production bulk composition.

The term “production bulk composition” as used herein refers to thecomposition existing prior to filling of the composition into individualdosing units.

In one embodiment of the instant invention the hydrophilic polymeremployed is hyaluronic acid and the detergent employed is Polysorbate80.

In one further embodiment of the present invention the hydrophilicpolymer employed is hyaluronic acid and the detergent employed isPolysorbate 20.

In one further embodiment of the present invention the hydrophilicpolymer employed is polyvinylpyroldone (PVP) and the detergent employedis Polysorbate 80.

The formulation of the invention is free of stabilising proteins.

The term “free of stabilising proteins” within the meaning of thepresent invention refers to formulations being free of peptides orproteins that stabilize the active peptide or protein. Examples for suchexcipients are, but not limited to, human serum albumin (HSA), gelatine,amino acids such as histidine, lysine, methionine or immunoglobulins.

The formulation of the instant invention is used for stabilisingproteins, peptides, or mixtures thereof.

The present invention further pertains to a composition comprising saidformulation and an active agent which may be a protein, a peptide,naturally occurring or modified/artificial or a mixture thereof

The term “stable composition” as used herein relates to a composition,wherein the protein or peptide retains upon storage for at least 4 weeksat room temperature, 60% relative humidity (RH) its physical andchemical stability and integrity up to 50%, 60%, 70%, 80% and 90%compared to the value measured after lyophilisation, meaning prior tostorage.

In a further embodiment, the composition of the invention is composedsuch that the protein or peptide retains upon storage for at least 6months at room temperature, 60% RH its physical and chemical stabilityand integrity up to 50%, 60%, 70%, 80% and 90% compared to the valuemeasured after lyophilisation, meaning prior to storage.

In a still further embodiment, the composition of the invention iscomposed such that the protein or peptide retains upon storage for atleast 12 months at room temperature, 60% RH its physical and chemicalstability and integrity up to 50%, 60%, 70%, 80% and 90% compared to thevalue measured after lyophilisation, meaning prior to storage.

As to the biological activity, “stable composition” refers to acomposition, wherein the neurotoxic component in a reconstituted oraqueous solution of pharmaceutical composition has greater than about20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and up to about 100% of thetoxicity that the biologically active neurotoxic component had prior tobeing incorporated into the pharmaceutical composition.

The term “room temperature” also designated as RT (or ambienttemperature) within the meaning of the instant invention, refers to thedefinition of U.S Pharmacopeia as being 20-25° C. (68-77° F.).

The term “relative humidity” also designated as RH within the meaning ofthe instant invention, refers to the ratio of the amount of water vaporin the air at a specific temperature to the maximum amount that the aircould hold at that temperature, expressed as a percentage.

In one aspect of the invention said composition herein is stable for 7months at 25° C. and 60% RH in lyophilised form. In another aspect ofthe invention said composition is stable for 3 months at 25° C. and 60%RH in lyophilised form. In one further aspect of the invention saidcomposition is stable for 2 months at 25° C. and 60% RH in lyophilisedform. In another aspect of the invention said composition is stable for1 month at 25° C. and 60% RH in lyophilised form.

In another aspect of the invention said composition is stable for 7months at 40° C. and 75% RH as in lyophilised form. In one furtheraspect of the invention said composition is stable for 3 months at 40°C. and 75% RH in lyophilised form. In one further aspect of theinvention said composition is stable for 2 months at 40° C. and 75% RHin lyophilised form. In one further aspect of the invention saidcomposition is stable for 1 month at 40° C. and 75% RH in lyophilisedform.

In one embodiment of the instant invention the stability is measured bymeasuring the extent of aggregation as a function of time as anindicator of protein stability. In another embodiment, stability of theprotein composition may be measured using the analytical methods knownto the one skilled in the art by determining % of intact protein, e.g.proteolytic cleavage, cell based assay. In one further embodiment thestability of the protein composition was determined by employing aMouse-hemidiaphragm assay (HDA-assay). In one embodiment of the instantinvention HDA-assay is employed to determined the stability of thecompositions claimed herein. The results are demonstrated as the potencymeasured in an HDA assay.

The HDA-Assay is conducted as defined by Göschel et al. (“BotulinumToxin Therapy: Neutralizing and Nonneutralizing Antibodies—TherapeuticConsequences” Experimental Neurology, 1997; 147: 96-102).

The instant invention further pertains to a composition that comprisesthe said formulation and a peptide, a protein or a mixture thereof,being naturally occurring or modified/artificial. Modification compriseschemical modification e.g. by glycosylation, acetylation, acylation orthe like, which may be beneficial e.g. to the uptake or stability of theprotein. The polypeptide chain of the protein may, however,alternatively or additionally be modified by addition, substitution ordeletion of one or more amino acid residues.

The term “peptide” within the meaning of the present invention refers toshort polymers formed by linking in a defined order of alpha-aminoacids.

The term “protein” as used herein relates to compounds of amino acidsarranged in a linear chain and joined together by peptide bonds betweenthe carboxyl and amino groups of adjacent amino acid residues. The term“protein” is used synonymously with the term “polypeptide”. Proteinsaccording to the instant invention may be artificial or naturallyoccurring.

The active protein or peptide in the formulation claimed herein may beartificial/modified or naturally occurring.

The term “artificial protein” within the meaning of the presentinvention refers to modified proteins. The term “modified protein”encompasses all possible modifications known to the person skilled inthe art, e.g. chemical modification, deletion.

The term “naturally occurring” within the meaning of the presentinvention refers to proteins or peptides found naturally in mammalorganism.

In one embodiment of the present invention, said protein is selectedfrom the group consisting of toxins, chondroitin, elastin, actin,myosin, aprotinin, growth hormone, growth hormone releasing factor,parathyroid hormone, thyroid stimulating hormone, lipoproteins (LDL,IDL, VLDL, VHDL, HDL), apolipoproteins (ApoA-1, ApoA-II, ApoA-IV,ApoC-I, ApoC-II, ApoC-III, ApoD, ApoE), α-1 Antitrypsin, insulin,proinsullin, follicle stimulating hormone, calcitonin, oxytocin,vasopressin, leuprolide acetate, somatostatin, luteinizing hormone,glucagons, clotting factors, anti-clotting factors, plasminogenactivator, human macrophage inflammatory protein, vascular endothelingrowth factor (VEGF), rheumatoid factors, bone derived neurotrophicfactor (BDNF), nerve growth factor-β (NGF-β), platelet-derived growthfactor (PDGF), fibroblast growth factor (FGF), epidermal growth factor(EGF), transforming growth factor (TGF-β1, TGF-β2, TGF-β3, TGF-β4,TGF-β5), erythropoietin, interleukins (IL-1 to IL-10), bone morphogenicprotein (BMP) parathyroid hormone, DNAse, cationic ferritin, interferon(α, β, γ) and mixtures thereof.

In another embodiment of the present invention said protein is a toxin.In one further embodiment of the instant invention said toxin is, abotulinum toxin, a diphtheria toxin or a tetanus toxin, or a mixture oftwo or more thereof.

In one embodiment of the present invention the protein in the saidcomposition is botulinum toxin.

In one further embodiment of the instant invention said botulinum toxinis selected from the group consisting of type A, B, C, C₁, D, E, F andG. In another embodiment of the present invention said botulinum toxinis type A. In one further embodiment of the instant invention saidprotein is the neurotoxic component of botulinum toxin type A.

The term “botulinum toxin” as used throughout the present application,refers to the neurotoxic component devoid of any other clostridialproteins, but also to the “botulinum toxin complex”. The term “botulinumtoxin” is used herein in cases when no discrimination between the toxincomplex and the neurotoxic component is necessary or desired. “BoNT” or“NT” are commonly used abbreviations.

The “neurotoxic component” of the botulinum toxin complex is initiallyformed as a single polypeptide chain, having in the case of serotype A amolecular weight of approximately 150 kDa. In other serotypes theneurotoxic component has been observed to vary between about 145 andabout 170 kDa, depending on the bacterial source. In the case ofserotype A, for example, proteolytic processing of the polypeptideresults in an activated polypeptide in the form of a dichain polypeptideconsisting of a heavy chain and a light chain, which are linked by adisulfide bond. In humans, the heavy chain mediates binding topre-synaptic cholinergic nerve terminals and internalization of thetoxin into the cell. The term “neurotoxic component” also includesfunctional homologs found in the other serotypes of Clostridiumbotulinum. In one embodiment of the present invention, the neurotoxiccomponent is devoid of any other C. botulinum protein, e.g. also devoidof RNA, which might potentially be associated with the neurotoxiccomponent. The neurotoxic component may be the single chain precursorprotein of approximately 150 kDa or the proteolytically processedneurotoxic component, comprising the light chain (Lc) of approximately50 kDa and the heavy chain (Hc) of approximately 100 kDa, which may belinked by one or more disulfide bonds (for a review see e.g. Simpson LL, Ann Rev Pharmacol Toxicol. 2004; 44:167-93). In humans, the heavychain mediates binding to pre-synaptic cholinergic nerve terminals andinternalization of the toxin into the cell. The light chain is believedto be responsible for the toxic effects, acting as zinc-endopeptidaseand cleaving specific proteins responsible for membrane fusion (SNAREcomplex) (see e.g. Montecucco C., Shiavo G., Rosetto O: The mechanism ofaction of tetanus and botulinum neurotoxins. Arch Toxicol. 1996; 18(Suppl.): 342-354)).

The neurotoxic subunit of the botulinum toxin complex is referred inthis document as the “neurotoxic component” or the “neurotoxic componentfree of complexing proteins”. The production of the neurotoxic componentof botulinum toxin type A and B are described, for example, in theinternational patent application WO 00/74703.

In a further embodiment the botulinum toxin is botulinum toxin type A.In one embodiment said botulinum toxin is free of any complexingproteins (neurotoxic component). In one further embodiment it is thepure neurotoxic component serotype A. In addition thereto, modified aswell as recombinant produced neurotoxic components of botulinum toxinsincluding the respective mutations, deletions, etc. are also within thescope of the present invention. With respect to suitable mutants,reference is made to WO 2006/027207 A1, WO 2009/015840 A1, WO2006/114308 A1 and EP 08015287.9 which are fully incorporated byreference herein. Furthermore, within the present invention, mixtures ofvarious serotypes (in the form the neurotoxic component or recombinantform or both forms thereof, e.g. mixtures of botulinum neurotoxins oftypes A and B) may be used. The present invention, however, also refersto toxins, e.g. botulinum toxins, which are chemically modified, e.g. bypegylation, glycosylation, sulfatation, phosphorylation or any othermodification, in particular of one or more surface or solvent exposedamino acid(s). Such botulinum toxins are disclosed in e.g. EP 08015288.7and the prior art disclosed therein.

In accordance with the teaching of the present invention, it alsoencompasses that the medicament contains no proteins found in thebotulinum toxin complex other than the neurotoxic component.

The botulinum toxin, preferably the neurotoxic component referred toherein, may be the sole active component or may contain additionalpharmaceutically active components.

In one embodiment the composition is lyophilized.

In one embodiment of the instant invention, the liquid compositions canbe filled into lyo-vials and subsequently lyophilized. Lyophilisation ofthe samples is conducted by freezing the samples at temperatures between−35° C. to −65° C. for a period of from 1 to 10 hours, e.g. 5 to 10hours. This step is followed by primary drying at a shelf temperature of−30° C. to 10° C., e.g. −20° C. to 10° C. or 5° C. to 10° C. under apressure of 100 mTorr to 200 mTorr for a period of 10 hours to 25 hours.Finally, the samples enter the last step of the lyophilisation process,being secondary drying, which is conducted at a shelf temperature of 15°C. to 25° C. for 5 hours to 15 hours. Sample volume in the lyo-vialsvaries between 0.1 to 5 ml, e.g. 0.2 to 1 ml or 0.4 to 0.6 ml, or 0.5ml. In one embodiment sample volume is between 2 ml to 4 ml.

In one further embodiment of the present invention, the lyophilisationprocess can be conducted by freezing the samples at a shelf temperatureof −45° C. for about 2 hours followed by primary drying at a shelftemperature of −25° C. and 90 mTorr for 12 hours, and secondary dryingat a shelf temperature of 25° C. for 12.5 hours.

In one embodiment an injectable solution comprising the said compositionis claimed.

The injectable solution claimed herein is stable at a temperature of 2to 8° C. for 24 hours.

In one embodiment said injectable solution is obtained by reconstitutingsaid lyophilised composition with a pharmaceutically acceptable sterilesolvent prior to administration to a mammal.

In one further embodiment, the present invention relates to a processfor the preparation of said injectable solution designed forintravenous, subcutaneous, intramuscular, intra-articular,intraperitoneal, intracerobrospinal, intracardiac, intrathecal,intravesical, intraosseous, intravitreal, epidural, intrasynovialinjection into a mammal. Said process comprises the step of dissolvingthe said lyophilised composition as claimed herein, prior toadministration, in a pharmaceutical acceptable sterile solvent.

In another embodiment of the instant invention, said injectable solutionis also administered via other routes of administration. Such routes ofadministration are, but not limited to, inhalation, oral and nasal. Anexample for such an application is, but not limited to, for instance,inhalation of α-1 Antitrypsin by COPD patients in form of an injectablesolution as claimed herein.

The composition as claimed herein is for use as a medicament, a cosmeticproduct, a cosmoceutical product or a diagnostic product.

The term “medicament” as used herein relates to a product or a mixtureof products, wherein said products may be mixed prior to administrationor be used one after another and have a therapeutic and/or diagnosticoutcome on the mammal they are administered to.

The term “cosmetic product” as used herein relates to products employedfor cosmetic purposes. The term “cosmetic” as used herein relates toproducts as defined in the FD&C Act, sec. 201(i) (Federal Food, Drug andCosmetic Act, FDA) as intended to be rubbed, poured, sprinkled, orsprayed on, introduced into, or otherwise applied to the human body forcleansing, beautifying, promoting attractiveness, or altering theappearance”.

The term “diagnostic product” as used herein relates to any productcomprising any compound or compounds that is delivered to a patient inorder to carry out a diagnostic test or assay on the patient.

The term “cosmeceutical product” as used herein relates to anon-prescription cosmetic product, that has also medicinal or drug-likebenefits.

In one embodiment of the present invention the claimed formulationherein may comprise a buffer.

The term “buffer” as used herein relates to an aqueous solutionconsisting of a mixture of a weak acid and its conjugate base or a weakbase and its conjugate acid.

In one further embodiment of the present invention the buffer isselected from the group consisting of phosphate buffer, acetate buffer,citrate buffer, formate buffer, benzoate buffer, TRIS(Tris(hydroxymethyl)-aminomethan) and maleate buffer. Said buffer isprepared according to the specifications of USP (United StatesPharmacopoeia), EP (European Pharmacopoeia) and the JP (JapanesePharmacopoeia) by using Pharmacopoeia-conform excipients. The bufferconcentration is to be determined in regard to the pH of the endproduct.

The excipients and the actives (peptides and/or proteins) employed inthe formulation herein are pharmaceutically acceptable.

The term “pharmaceutically acceptable” as used herein relates to anyexcipient, pharmaceutically active ingredient, which enables the saidcomposition to be taken by mammals at therapeutically effectiveconcentration, avoiding any kind of side effects.

In one aspect the present invention pertains to a kit comprising one ormore containers comprising the formulation/composition and instructionsfor use of the formulation/composition and optionally a pharmaceuticallyacceptable sterile solvent.

The term “solvent” as used herein relates to any liquid which aids indissolving or diluting any other substance or substance mixture or aproduct. The term “solvent” within the meaning of the instant inventionmay encompass also a mixture of solvents.

The pharmaceutical acceptable sterile solvent to be employed within saidprocess is, but not limited to, water for injection (WFI), isotonic saltsolution, Ringer's solution, pH-buffered solution, an aqueous solutionof 5% glucose.

A further aspect of the present invention relates to a sterilecomposition.

The term “sterile” as used herein relates to the absence of undesiredmicroorganisms and relates to the norms defined in the USP (UnitedStates Pharmacopoeia), EP (European Pharmacopoeia) and the JP (JapanesePharmacopoeia).

In one embodiment the composition is non-pyrogenic e.g. containing <1 EU(endotoxin unit, a standard measure) per dose, and preferably <0.1 EUper dose. In one further embodiment said injectable solution is alsosterile and non-pyrogenic.

In one embodiment of the instant invention said composition is for usein vertebrates, such as mammals.

The term “vertebrate” is defined herein as any member of the subphylumvertebrata, chordates with backbones or spinal columns. Therefore theterm “vertebrate” encompasses humans, mammals, marsupials, reptiles,birds, amphibians and fish.

The term “mammal” in this document is defined as any warm-blooded,vertebrate characterized by the presence of sweat glands, including milkproducing glands, and by the presence of hair, three middle ear bonesused in hearing, and a neocortex region in the brain. A male or femalehuman, dog, cat, pig, cow, horse, donkey, sheep, goat and deer istherefore encompassed by this definition of a mammal.

The term “marsupial” is defined herein as a mammal in which the femaletypically has a pouch in which it rears its young through early infancy.They differ from placental mammals in their reproductive traits.

The term “reptile” is defined herein as any air-breathing, ectothermicvertebrate that has skin covered in scales as opposed to hair orfeathers.

The term “bird” is defined herein as any bipedal, warm-blooded,vertebrate that lays eggs.

The term “amphibian” is defined herein as all living tetrapods(four-legged vertebrates) that do not have amniotic eggs, areectothermic and generally spend part of their time on land.

The term “fish” is defined herein as aquatic vertebrates that aretypically ectothermic, covered with scales, and equipped with two setsof paired fins and several unpaired fins.

The concentration values herein are expressed in “about” values.

The term “about” as used herein is intended to reflect a variation of20% of the value it is attached to.

The instant invention further relates to a process for preparing thesaid composition characterized in that said composition is prepared asan aqueous composition and subsequently lyophilized.

Prior to lyophilisation, the protein or peptide is dissolved in anaqueous solution, which is stabilized by a hydrophilic polymer, amixture of polyalcohol and a sugar, a detergent. The stabilization ofthe protein in solution means that the protein is enveloped by astructure composed of hydrophilic polymer, a detergent and a mixture ofpolyalcohol and sugar.

By using a detergent, it is possible to reduce the amount of hydrophilicpolymers. In one embodiment by using Tween 80 the concentration of PVPwas reduced from 150 mg/g to 80 mg/g based on the total weight ofproduction bulk composition. Due to such an effect, the industrialproduction of the composition herein was improved.

In one embodiment of the present invention the composition hereincomprises the neurotoxic component of botulinum toxin in a quantity ofabout 2 pg to 50 ng per 1 g production bulk composition. Preferredquantity ranges are in the range of from 2 pg to 200 pg, 200 pg to 400pg, 400 pg to 600 pg, 600 pg to 800 pg, 800 pg to 1 ng, 1 ng to 1.5 ng,1.5 ng to 2 ng, 2 ng to 2.5 ng, 2.5 ng to 3 ng, 3 to 3.5 ng, 3.5 to 4ng, 4 ng to 4.5 ng, and 4.5 to 5 ng per 1 g of water, respectively per 1g production bulk composition. In an embodiment of the instantinvention, the neurotoxic component has a biological activity of 50 to250 LD₅₀ units per ng neurotoxic component, as determined in a mouseLD₅₀ assay. In one further embodiment, the neurotoxic component has abiological activity of about 150 LD₅₀ per ng neurotoxic component.

The following demonstrates some embodiments of the stable compositionsas claimed herein, wherein the amounts of the constituents specified areall relative to 1 g production bulk composition.

In one further embodiment of the present invention, the compositionclaimed herein comprises ≦1.6 ng neurotoxic component of botulinumtoxin, about 0.5 mg of hyaluronic acid, about 15.0 mg of mannitol, about5.0 mg of sucrose and about 0.1 mg of Polysorbate 80.

In another embodiment of the present invention, the composition claimedherein comprises ≦1.6 ng neurotoxic component of botulinum toxin, about1.0 mg of hyaluronic acid, about 30.0 mg of mannitol, about 10.0 mg ofsucrose and about 0.2 mg of Polysorbate 80.

In another embodiment of the present invention, the composition claimedherein comprises ≦1.6 ng neurotoxic component of botulinum toxin, about2.0 mg of hyaluronic acid, about 40.0 mg of mannitol, about 10.0 mg ofsucrose and about 0.5 mg of Polysorbate 80.

In another embodiment of the present invention, the composition claimedherein comprises ≦1.6 ng neurotoxic component of botulinum toxin, about2.0 mg of hyaluronic acid, about 40.0 mg of mannitol, about 10.0 mg ofsucrose and about 0.2 mg of Polysorbate 80.

In one further embodiment of the present invention, the compositionclaimed herein comprises ≦1.6 ng neurotoxic component of botulinumtoxin, about 1.0 mg of hyaluronic acid, about 40.0 mg of mannitol, about10.0 mg of sucrose and about 0.5 mg of Polysorbate 80.

In one further embodiment of the present invention, the compositionclaimed herein comprises ≦1.6 ng neurotoxic component of botulinumtoxin, about 1.0 mg of hyaluronic acid, about 10.0 mg of sucrose andabout 0.2 mg of Polysorbate 80.

In one further embodiment of the present invention, the compositionclaimed herein comprises ≦1.6 ng neurotoxic component of botulinumtoxin, about 80.0 mg of polyvinylpyrollidone (PVP), about 50 mg ofmannitol, and about 0.2 mg of Polysorbate 80.

In one further embodiment of the present invention, the compositionclaimed herein comprises ≦1.6 ng neurotoxic component of botulinumtoxin, about 80.0 mg of polyvinylpyrollidone (PVP), about 50 mg ofmannitol, and about 0.5 mg of Polysorbate 80.

In one further embodiment of the present invention, the compositionclaimed herein comprises ≦1.6 ng neurotoxic component of botulinumtoxin, about 100.0 mg of polyvinylpyrollidone (PVP), about 30.0 mg ofmannitol, about 10.0 mg of sucrose and about 0.2 mg of Polysorbate 80.

In one further embodiment of the present invention, the compositionclaimed herein comprises ≦1.6 ng neurotoxic component of botulinumtoxin, about 80.0 mg of polyvinylpyrollidone (PVP), about 30.0 mg ofmannitol, about 10.0 mg of sucrose and about 0.2 mg of Polysorbate 80.

In one further embodiment of the present invention, the compositionclaimed herein comprises ≦1.6 ng neurotoxic component of botulinumtoxin, about 80.0 mg of polyvinylpyrollidone (PVP), about 30.0 mg ofmannitol, about 10.0 mg of sucrose about 0.2 mg of Polysorbate 80 andabout 10 mM phosphate buffer (pH 7.4).

In one further embodiment of the present invention, the compositionclaimed herein comprises ≦1.6 ng neurotoxic component of botulinumtoxin, about 50.0 mg of polyvinylpyrollidone (PVP), about 30.0 mg ofmannitol, about 10.0 mg of sucrose about 0.2 mg of Polysorbate 80 andabout 10 mM phosphate buffer (pH 7.4).

In one further embodiment of the present invention, the compositionclaimed herein comprises ≦1.6 ng neurotoxic component of botulinumtoxin, about 100.0 mg of polyvinylpyrollidone (PVP), about 30.0 mg ofmannitol, about 10.0 mg of sucrose and about 0.5 mg of Polysorbate 80.

In one further embodiment of the present invention, the compositionclaimed herein comprises ≦1.6 ng neurotoxic component of botulinumtoxin, about 100.0 mg of polyvinylpyrollidone (PVP), about 50.0 mg ofmannitol, and about 0.2 mg of Polysorbate 80.

In one further embodiment of the present invention, the compositionclaimed herein comprises ≦1.6 ng neurotoxic component of botulinumtoxin, about 100.0 mg of polyvinylpyrollidone (PVP), about 50.0 mg ofmannitol, and about 0.5 mg of Polysorbate 80.

In one further embodiment of the present invention, the compositionclaimed herein comprises ≦1.6 ng neurotoxic component of botulinumtoxin, about 150.0 mg of polyvinylpyrollidone (PVP), about 30.0 mg ofmannitol, about 10.0 mg of sucrose and about 0.2 mg of Polysorbate 80.

Composition as claimed herein is for treatment of a disease or conditioncaused by or associated with hyperactive cholinergic innervation ofmuscles or exocrine glands in a patient, where the neurotoxic componentblocks acetylcholine secretion into the synaptic cleft. Therefore, thecomposition claimed by the present invention may be directed to thetreatment of any of the following indications, most of which aredescribed in detail in Dressler D (2000) (Botulinum Toxin Therapy.Thieme Verlag, Stuttgart, New York):

dystonia    cranial dystonia       blepharospasm       oromandibulardystonia          jaw opening type          jaw closing type      bruxism       Meige syndrome       lingual dystonia       apraxiaof eyelid opening    cervical dystonia       antecollis      retrocollis       laterocollis       torticollis    pharyngealdystonia    laryngeal dystonia       spasmodic dysphonia/adductor type      spasmodic dysphonia/abductor type       spasmodic dyspnea    limbdystonia       arm dystonia          task specific dystonia            writer's cramp             musician's cramps            golfer's cramp       leg dystonia          thigh adduction,thigh abduction          knee flexion, knee extension          ankleflexion, ankle extension          equinovarus deformity       footdystonia          striatal toe          toe flexion          toeextension       axial dystonia          pisa syndrome          bellydancer dystonia       segmental dystonia       hemidystonia      generalised dystonia    dystonia in lubag    dystonia incorticobasal degeneration    dystonia in lubag    tardive dystonia   dystonia in spinocerebellar ataxia    dystonia in Parkinson's disease   dystonia in Huntington's disease    dystonia in Hallervorden Spatzdisease    dopa-induced dyskinesias/dopa-induced dystonia    tardivedyskinesias/tardive dystonia    paroxysmal dyskinesias/dystonias      kinesiogenic       non-kinesiogenic       action-induced palatalmyoclonus myoclonus myokymia rigidity benign muscle cramps hereditarychin trembling paradoxic jaw muscle activity hemimasticatory spasmshypertrophic branchial myopathy maseteric hypertrophy tibialis anteriorhypertrophy nystagmus oscillopsia supranuclear gaze palsy epilepsiapartialis continua planning of spasmodic torticollis operation abductorvocal cord paralysis recalcitant mutational dysphonia upper oesophagealsphincter dysfunction vocal fold granuloma stuttering Gilles de laTourette syndrom middle ear myoclonus protective larynx closurepostlaryngectomy speech failure protective ptosis entropion sphincterOdii dysfunction pseudoachalasia nonachalsia oesophageal motor disordersvaginismus postoperative immobilisation tremor bladder dysfunction   detrusor sphincter dyssynergia    bladder sphincter spasm hemifacialspasm reinnervation dyskinesias cosmetic use    craw's feet    frowning   facial asymmetries    mentalis dimples stiff person syndrome tetanusprostate hyperplasia adipositas treatment infantile cerebral palsystrabismus    mixed    paralytic    concomitant    after retinaldetachment surgery    after cataract surgery    in aphakia    myositicstrabismus    myopathic strabismus    dissociated vertical deviation   as an adjunct to strabismus surgery    esotropia    exotropiaachalasia anal fissures exocrine gland hyperactivity    Frey syndrome   Crocodile Tears syndrome    hyperhidrosis       axillar       palmar      plantar rhinorrhea relative hypersalivation       in stroke      in parkinsosn's       in amyotrophic lateral sclerosis spasticconditions       in encephalitis and myelitis          autoimmuneprocesses             multiple sclerosis             transverse myelitis            Devic syndrome          viral infections          bacterialinfections          parasitic infections          fungal infections      in hereditary spastic paraparesis    postapoplectic syndrome      hemispheric infarction       brainstem infarction       myeloninfarction    in central nervous system trauma       hemispheric lesions      brainstem lesions       myelon lesion    in central nervous systemhemorrhage       intracerebral hemorrhage       subarachnoidalhemorrhage       subdural hemorrhage       intraspinal hemorrhage    inneoplasias       hemispheric tumors       brainstem tumors       myelontumors

In another embodiment, the present invention pertains to a kit, whereinsaid kit comprises one or more of a container comprising theformulation/composition claimed herein, instructions for reconstitutingthe said formulation/composition and optionally, a pharmaceuticallyacceptable sterile solvent. Suitable containers include, but are notlimited to, single vials, dual chamber vials, single applicationsyringes or dual chamber syringes. The container may be formed from avariety of material such as glass or plastic adapted for pharmaceutical,diagnostic, cosmetic or cosmeceutical administration. The said kit maybe adapted for single use or for multiple uses.

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention should not be construed as being limited to these examples,but rather should be construed to encompass any and all variations whichbecome evident as a result of the teaching provided herein. Thefollowing materials and methods are provided with respect to thesubsequent examples but do not limit a multiplicity of materials andmethodologies encompassed by the present invention.

Examples

Studies were conducted to find a stabilized composition of botulinumtoxin type A. Each composition comprised of ≦1.6 ng neurotoxic componentof botulinum toxin type A. The composition of the screening formulationsis summarized in the following table, wherein the amounts are given asmg per 1 g of the production bulk composition.

PVP Hyaluronic acid Mannitol Sucrose Polysorbate 80 Formulation No[mg/g] [mg/g] [mg/g] [mg/g] [mg/g] Comparative 100 — 50 — 0.5Formulation 1 Comparative — 1 30 10 — Formulation 2 Comparative — — 3010 0.2 Formulation 3 Example 1  80 — 30 10 0.2 Example 2 — 2 40 10 0.5Example 3 — 2 30 10 0.2 Example 4 — 1 30 10 0.2 Start 40° C./75% RH 40°C./75% RH 40° C./75% RH Value 1 Month 3 Months 7 Months Formulation NoT½ [min] Comparative 68 76 98 n.m. Formulation 1 Comparative 138 110n.m. n.m. Formulation 2 Comparative 96 n.m. n.m. n.m. Formulation 3Example 1 67 72 87 90 Example 2 66 85 n.m. n.m. Example 3 69 78 n.m.n.m. Example 4 68 69 n.m. 67 Start 25° C./60% RH 25° C./60% RH 25°C./60% RH Value 1 Month 3 Months 7 Months Formulation No T½ [min]Example 1 67 69 75 n.m. Example 4 71 64 74 66 n.m. = not measured

The stability of the formulations were determined by using a HDA-assayas defined by Goschel et al. (“Botulinum Toxin Therapy: Neutralizing andNonneutralizing Antibodies—Therapeutic Consequences” ExperimentalNeurology, 1997; 147: 96-102). The start value was measured afterlyophilisation. The mean of all four experiments is calculated andcompared to the data generated by the reference material. The change ofparalysis time over time of storage indicates the stability of theneurotoxin formulation: increasing of sample paralysis time compared tooriginal values indicate a loss of activity of neurotoxin.

The results are expressed in minutes required to reach half of theinitial muscle concentration force. Shorter time valves reflect higheramounts of active toxin. The results can be interpreted as follows: Inprinciple the formulations according to the present invention are morestable compared to the Comparative Formulations, wherein one of theconstituents required is missing.

It becomes apparent from Comparative Formulation 1, in the absence ofsucrose, a formulation with an active toxin may be prepared, which,however, looses rapidly its activity such that within 3 months at 40° C.and 75% RH the activity of the toxin is significantly reduced.Comparative Formulation 2 shows that in absence of the detergent, thetoxin significantly looses activity already during production, asbecomes apparent from the high starting value of 138 min directlymeasured after lyophilization. Comparative Formulation 3 shows that inabsence of the hydrophilic polymer, the toxin significantly loosesactivity already during production, as becomes apparent from the highstarting value of 96.

1. A formulation comprising (a) a hydrophilic polymer, (b) a mixture ofa polyalcohol and a sugar, wherein the weight ratio of polyalcohol tosugar is from 2:1 to 5:1 (wt-%), (c) a detergent, and wherein theformulation is free of stabilizing proteins.
 2. The formulation of claim1, wherein the hydrophilic polymer is selected from hyaluronic acid,polyvinylpyrollidone (PVP), copolymers of N-vinylpyrollidone, acellulose derivative, dextran, polyethyleneglycol (PEG), PEG/PPG blockcopolymers, homo- and copolymers of acrylic and methacrylic acid,polyurethanes, polyvinyl alcohol, polyvinylethers, maleic anhydridebased copolymers, polyesters, vinylamines, polyethyleneimines,polyethyleneoxides, poly(carboxylic acids), polyamides, polyanhydrides,polyphosphazenes, and mixtures thereof.
 3. The formulation of claim 2,wherein the cellulose derivative is selected from hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, methylcellulose and carboxymethyl cellulose.
 4. The formulation of claim 1,wherein the polyalcohol is selected from mannitol, inositol, lactilol,isomalt, xylitol, erythritol, sorbitol, and mixtures thereof.
 5. Theformulation of claim 1, wherein the sugar is selected frommonosaccharides, disaccharides, polysaccharides, and mixtures thereof.6. The formulation of claim 1, wherein the detergent is selected fromnon-ionic surfactants, anionic surfactants and cationic surfactants. 7.The formulation of claim 1, further comprising a peptide, a protein or amixture thereof.
 8. The formulation of claim 7, which is lyophilized. 9.The formulation of claim 7, wherein the protein is selected from toxins,chondroitin, elastin, actin, myosin, aprotinin, growth hormone, growthhormone releasing factor, parathyroid hormone, thyroid stimulatinghormone, lipoproteins (LDL, IDL, VLDL, VHDL, HDL), apolipoproteins(ApoA-1, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III, ApoD, ApoE), α-1Antitrypsin, insulin, proinsullin, follicle stimulating hormone,calcitonin, oxytocin, vasopressin, leuprolide acetate, somatostatin,luteinizing hormone, glucagons, clotting factors, anti-clotting factors,plasminogen activator, human macrophage inflammatory protein, vascularendothelin growth factor (VEGF), rheumatoid factors, bone derivedneurotrophic factor (BDNF), nerve growth factor-β (NGF-β),platelet-derived growth factor (PDGF), fibroblast growth factor (FGF),epidermal growth factor (EGF), transforming growth factor (TGF-β1,TGF-β2, TGF-β3, TGF-β4, TGF-β5), erythropoietin, interleukins (IL-1 toIL-10), bone morphogenic protein (BMP) parathyroid hormone, DNAse,cationic ferritin, interferon (α, β, γ) and mixtures thereof.
 10. Theformulation of claim 9, wherein the toxin is selected from a botulinumtoxin, diphtheria toxin, tetanus toxin and mixtures thereof.
 11. Theformulation of claim 1, which comprises Clostridium botulinum toxin,hyaluronic acid, mannitol, sucrose, polysorbate 80 and, optionally,water for injection.
 12. An injectable solution comprising theformulation of claim
 7. 13. The formulation of claim 7, in the form of acosmetic product, a cosmeceutical product or a diagnostic product. 14.The formulation of claim 10, comprising a therapeutically effectiveamount of the toxin for the treatment of a disease or condition causedby or associated with hyperactive cholinergic innervation of muscles orexocrine glands in a patient.
 15. A kit comprising (a) one or morecontainers comprising the formulation/composition of any of thepreceding claims, and (b) instructions for use of the formulation ofclaim 1, and optionally, (c) a pharmaceutically acceptable sterilesolvent.
 16. A method for stabilizing proteins, peptides, or mixturesthereof, comprising admixing the formulation of claim 1 with a protein,peptide, or mixtures thereof.